In the mammalian small intestine Na is primarily absorbed by coupled NaCl absorption and Na-glucose co-transport (SGLT-1) on the brush border membrane of villus cells. Coupled NaCl absorption occurs via the dual operation of Na:H (specifically NHE3) and Cl:HCO3 exchange. The regulation of NHE3 and SGLT-1 in the normal intestine by nitric oxide (NO), one of the most biologically active molecules, was unclear. We demonstrated that whether in vivo in rabbits and/or in vitro inhibition in rat intestinal epithelial cells, inhibition of NO inhibited villus cell BBM SGLT-1 and stimulated NHE3. The mechanism of inhibition of SGLT-1 was by reducing its affinity for glucose specifically by altering the glycosylation of the protein. In contrast, NHE3 was stimulated secondary to an increase in BBM transporter numbers by an increase in the transcription and synthesis of BBM NHE3. Having shown that inhibition of NO regulates NHE3 and SGLT-1, the next logical question is what happens during physiological increases in NO. In fact, results to date indicate that BBM NHE3 and SGLT-1 may be compensatorily regulated by NO to maintain cellular Na homeostasis. These observations led us to ask a most novel question -- can these two BBM transport proteins directly regulate one another? A phenomenon here to fore not described in intestinal transport physiology. Given this background, the overall hypothesis of this proposal is that the two primary Na absorptive pathways in the enterocyte BBM regulate one another via NO and/or directly to maintain cellular Na homeostasis. Thus, the overall aim of this proposal is to determine the compensatory reciprocal mechanism of regulation of intestinal epithelial cell BBM NHE3 and SGLT-1 by NO and/or directly. Specifically using a combination of in vivo and in vitro models, employing complementary physiological and molecular techniques with appropriate pharmacological agents and specific molecular reagents we will: 1. Elucidate the enhanced NO mediated mechanism of regulation of BBM NHE3 and SGLT-1 and 2. Determine the mechanism of direct regulation of BBM NHE3 and SGLT-1 by each other. This study will provide novel insight into whether the two primary Na absorptive pathways in the BBM of intestinal epithelial cells can directly regulate the functioning of each other and how NO mediates the compensatory reciprocal regulation of NHE3 and SGLT-1. Na assimilation is essential to maintain health and it is critical for a wide range of common diseases such as diarrhea and hypertension. Similarly, glucose absorption is not only essential because carbohydrates represent the predominant nutrient in the diet, but also because it is important in a variety of common diseases from diabetes to obesity. In conclusion, the unique and direct regulation of the two primary Na absorptive pathways as hypothesized in this proposal could form the basis for developing strategies to promote electrolyte and nutrient absorption where deficient and inhibit the same in disease states where it would be advantageous. PUBLIC HEALTH RELEVANCE: Sodium and glucose assimilation in the intestine is essential to maintain good health. Their absorption is even more critical in numerous common diseases including diarrhea, diabetes, obesity and hypertension. Nitric oxide, one of the most biologically active molecules, may mediate the regulation of the absorption of sodium and glucose. In fact, our studies show that transporters primarily responsible for sodium and glucose absorption (SLGT-1 and NHE3) may directly regulate one another. Better understanding of the unique and direct regulation of these two primary Na absorptive pathways as hypothesized in this proposal could form the basis for developing strategies to promote electrolyte and nutrient absorption where deficient and inhibit the same in disease states where it would be advantageous.